Electron counting detectors in scanning transmission electron microscopy via hardware signal processing

Transmission electron microscopy is a pivotal instrument in materials and biological sciences due to its ability to provide local structural and spectroscopic information on a wide range of materials. However, the electron detectors used in scanning transmission electron microscopy are often unable to provide quantified information, that is the number of electrons impacting the detector, without exhaustive calibration and processing. This results in arbitrary signal values with slow response times that cannot be used for quantification or comparison to simulations. Here we demonstrate and optimise a hardware signal processing approach to augment electron detectors to perform single electron counting.

Full detector imperfection measurements Detector imperfections measured for 8 commercially available electron detectors from 5 manufacturers. Analog and counted maps were acquired simultaneously. Note that the ellipticity for detector D (marked with a †) uses the outer radius where all other measurements use the inner radius.

Low dose temporal response
The slow temporal response in analog imaging is clearly seen in low dose images, where the number of electron detection events is below 1 per pixel. Such an example images is shown in Fig. S2 with both analog and counted acquisition. The electron pulse shape of Fig. 1d in the main text is clearly visible across multiple pixels. Figure S2 Low dose pulse streaking a Single frame analog image a the cell wall of a human macrophage showing the same region as Fig. 4b in the main text. Image is 256×512 pixels with 50 ns per pixel. b Counted image acquired simultaneously to a. c, d Zoomed in regions of a and b, respectively, taken from the bottom right corner of each image.

Noise rejection
Depending on the detector, control electronics, and environment, the detector signal quality may be degraded. For lower quality signals, it is possible to erroneously detect noise as electrons. An example is shown in Fig. S3 where a relatively well-defined pulse (Fig. S3a) gives a gradient signal that can be thresholded such that 2 electrons can be detected (inset in Fig. S3b). A simple yet effective approach to reject signal noise is to require that the thresholding of the signal gradient must remain above the threshold for a set period before an electron is registered. Typically, this is only required to be 16 or 24 ns (2 or 3 samples for the hardware shown here). This also has the benefit that the digital output when an electron is detected is not significantly delayed. It is possible to choose a time step to calculate the gradient to further reject noise. For example, when sampling the signal at a high frequency, the difference between neighbouring samples on a rising edge is often not significantly above the noise level, however, across a number of samples the rising edge is clear. The gradient step should be tuned to the specifics of each detector system such that the signal gradient is clear, but rapid events can be distinguished. Figure S4 shows an example data stream with 3 clear pulses. By increasing the gradient step ( Fig.  S4b) noise can be reduced, but also the gradient signal can be attenuated. Increasing the gradient step also increase the lag between the actual electron detection event and the digital output, so should be as small as possible.

Sample preparation Human monocyte
For TEM, Human monocyte derived macrophage cells (HMMs) were grown on 6-well tissue culture plates, incubated with graphene (at a concentration of 10µg/ml) for 4 hours in SFM culture medium at 37 °C. After the incubation period, cell monolayers were washed twice in 0.9 % saline to remove any non-ingested particles and subsequently fixed with 4 % glutaraldehyde (in 0.1 M HEPES buffer, pH 7.2) for 1 h at 4 °C. Cells were scraped using a cell scraper and washed several times in deionized water (DIW) to remove fixative. Then, samples were osmicated (1% OsO4, 0.15 % potassium ferricyanide; 2 mM CaCl2 in DIW) for 1 h at RT. Again, samples were washed several times with DIW and then bulk stained for 1 h at RT in the dark using uranyl acetate. Following 2 washes in DIW, the samples were dehydrated in graded solutions of ethanol (70 %, 95 %, 100 %), 3x in each for 5 minutes, respectively. After two additional washes in 100 % acetonitrile, samples were infiltrated with Quetol 651 resin over 5 days using fresh resin each day. Resin was cured at 65 °C for 48h. Ultrathin sections (~ 70nm) were cut using a Leica Ultracut ultramicrotome, mounted on 300 mesh bare copper grids.

LaFeO3
The lanthanum ferrite ceramic was prepared from La2O3 and Fe2O3 powder through a solid-state reaction process. Further information is presented by A. Mostaed et al. (2021). 1 To achieve electron transparency, samples were thinned using Ar ions in a Gatan PIPS.

Gold nanoparticles
We used high resolution combined test specimen containing gold particles on carbon film purchased from Agar Scientific.

SrTiO3
(100) oriented SrTiO3 substrates were commercially purchased, from which a TEM lamella sample was prepared. A Tescan Amber focussed ion beam (FIB) was used with standard FIB lift-out techniques.